In the last decade, enormous progress in recombinant DNA technology has enabled the discovery and/or production of a large number of physiologically active proteins, peptides, enzymes, and genes, many of them having unforeseen potential to be used as pharmaceuticals.
Use of these proteins and peptides as medicines, however, suffers from many problems. First, peptides or proteins are very low in vivo absorption efficiency because they are easily hydrolyzed or degraded by enzymes within a short period of time after being taken into the body. Further, many pharmaceutically relevant peptides and proteins, even those having human primary structure, can be immunogenic, giving rise to production of neutralizing antibodies circulating in the blood stream. In addition, the clearance attributable to the reticuloendothelial system (RES) is high. Therefore, most protein and peptide drugs have been administered by injection, thus far. The administration by injection, however, causes the patients the pain and is accompanied by dangers. On the other hand, research in gene therapy has demonstrated potential for treatment of both acquired and inherited diseases. One of the major challenges for gene therapy is systemic delivery of a nucleic acid directly into an affected tissue. This requires development of a vehicle that is able to protect a nucleic acid from degradation, while delivering the genes of interest to specific tissues and target cell compartments. Non-viral gene delivery systems such as liposomes (H. M. Temin, J. Human Gene Therapy 111, 1990) or poly-L-lysine (PLL) (G. Y. Wu et al. 263 J. Biol. Chem. 14621, 1988) have drawbacks of low transfection efficiency or causing precipitation. Synthetic delivery systems also elicit fewer immunological complications with large scale repeated use (P. L. Felgner, 5 Adv. Drug Deliv. 163, 1990).
Conjugation of biologically active molecules, for example proteins or peptides, to synthetic macromolecules may afford great advantages when they are applied in vivo and in vitro. When being covalently bonded to macromolecules, biologically active molecules may exhibit modified surface properties and solubility, and thus may be increased in solubility within water or organic solvents. Further, the presence of macromolecules may make the conjugated proteins and peptides more stable in vivo as well as reduce the clearance by the intestines, the kidneys, the spleen, and/or the liver.
There have been many patents or publications regarding conjugation of biologically active molecules with polyethylene glycol (hereinafter, referred to as “PEG”) or similar water soluble polyalkylene oxides (hereinafter, referred to as “PAO”).
U.S. Pat. No. 4,179,337 discloses conjugates of biologically active polypeptides and PEG or polypropylene glycol (PPG) with a molecular weight of 500–20,000, which are water-soluble, biocompatible, biologically active, and non-immunogenic polymers. This patent discloses that the conjugation of PEG to proteins or peptides is achieved by reacting activated PEG to amino residues of proteins or peptides, lysine residues and N-termini. As for PEG activation, one of the hydroxyl groups of PEG is substituted with a methyl ether group while the other hydroxy group is bonded to an electrophilic functional group. Also it is described that PEG or PPG protects biologically active polypeptides from inactivation/denaturation.
U.S. Pat. No. 4,301,144 discloses the hemoglobin modified by conjugating hemoglobin with polyalkylene glycol or its derivatives. It is described therein that hemoglobin is increased in oxygen carrying potential and retention time in the body when being associated with PEG or water-soluble polymers.
Various proteins are reported to show extended half-lives and reduced immunogenicity in plasma when being conjugated with PEG (Abuchowski et al., Cancer Biochem. Biophys., 7, 175–186, 1984).
U.S. Pat. No. 5,951,974 and Algranati et al (Hepatology, 40 (suppl), 190A, 1999) describe that PEGylation of alpha interferon with PEG12000 as well as PEG40000 decreases the clearance rate, to achieve once-weekly subcutaneous injection instead of 3 times a week injection for native interferon.
Davis et al (Lancet, 2, 281–283, 1981) demonstrated that uricase-PEG conjugates had higher in vivo half-life and showed reduced side effects during the metabolism of uric acid.
Also, Niven et al (J. of Contr., Rel. 32, 177–189, 1994) demonstrated PEG conjugation of recombinant human granulocyte-colony stimulating factor (hereinafter, referred to as rhG-CSF) showed a more intense and extended white blood cell response relative to rhG-CSF alone.
However, there is a barrier to conjugating a number of linear polymers to proteins or peptides with retaining biological activity, because the active sites of proteins or peptides are spatially hindered. The conjugation of linear polymers with a molecular weight of 20,000 and higher has been attempted and resulted in the extended circulating half-life. The yield of this conjugate was, however, found to be very low and considered not to be economical.
To overcome the problem of conjugating linear polymer to proteins or peptides as mentioned above, the use of branched PEG has been attempted. U.S. Pat. No. 5,932,462 and U.S. Pat. No. 5,643,575 disclosed a branched or multi-armed aliphatic polymer derivative that is monofunctional and hydrolytically stable. The polymer arms are capped with relatively nonreactive end groups. The derivative can include a single reactive site that is located among the polymer moieties. However, these branched polymers with short length of linker between polymer and protein experience steric hindrance and thus reduce the reactivity and yield of product.
Also, U.S. Pat. No. 5,919,455 disclosed a branched aliphatic polymer derivative with various lengths of linkers including from 1 to 18 units of polyethylene glycol to improve the reactivity between polymer and protein. However, these branched PEG derivatives with a long linker including a PEG chain are too hydrophilic to use as efficient carriers for protein or genes.
Veronese et al. (Veronese, et al., Bioconjugate Chem, 12, 62, 2001) and WO 00/33881 introduced the preparation of branched PEGs with dipeptide as reporter to analyze the polymers easily and they showed different structures of polymers from the present invention.